1. Technical Field
The present invention relates generally to the field of stabilizing proteins, and more specifically to the field of stabilizing proteins without any modification of their primary sequence. The present invention further relates to stabilizing proteins by employing domain insertion of a target protein into a thermophilic scaffold protein.
2. Prior Art
High specificity and selectivity of a protein as a catalyst are of great importance in the (bio)chemical industry as these properties can reduce the number of reaction steps in synthesis and simplify product purification. For example, lipase has been employed for producing novel polymers that would otherwise be difficult to make by conventional chemical polymerization. However, despite a number of advantages over conventional chemical reactions, the progress of enzymatic reactions has been limited due to the insufficient stability of enzymes under common reaction conditions, such as the presence of organic solvents as well as high pressures and temperatures. Under these conditions, proteins unfold and lose activity significantly. In fact, limited stability is a common problem associated with most proteins.
Improvements in stability have been accomplished through rational, combinatorial and data-driven design. A large body of data has demonstrated that protein stabilization can be achieved by rational or combinatorial design or a combination of both. The rational design requires a knowledge of protein 3D structures and/or an understanding of forces and interactions affecting protein stability. Successful attempts have been reported in the rational design of highly stable proteins. Some rational protein stabilization strategies include “entropic stabilization” through rigidification by mutations, introduction of disulfide bridges, salt bridges, and clusters of aromatic-aromatic interactions, and engineering of subunit interfaces of multimeric proteins. Structural studies of extremophilic organisms and their proteins have provided significant insight into the molecular determinants of stabilization. Mesophilic proteins have been engineered to become highly stable through mutations found in corresponding thermophilic proteins. Comparative studies on a large number of naturally found or engineered stable proteins have revealed the existence of different ways of enhancing protein stability through mutations and recombinations. The combinatorial design requires construction of a diverse library and its screening to isolate variants with desired properties. A considerable amount of proteins with high stability have been identified using combinatorial design. Recently, data-driven design, where the library size is reduced by pinpointing specific residues to target based on structure and sequence information, has led to isolation of stable proteins.
Improvements in stability also have been accomplished by the addition of molecular chaperones and ligands. Molecular chaperones have been used not only for improving folding of a protein in vivo but also stability of a protein in vitro. Exposed hydrophobic surfaces, which should be buried in otherwise native protein structures, are the main targets of molecular chaperones. Addition of GroES, GroEL and ATP in vitro increased kinetic stability of alcohol dehydrogenase at 50° C. by two-fold. Similarly, the chaperone activity of aB-crystallin prevented unfolding and aggregation of citrate synthase at 45° C. Chemical chaperones, such as glycerol, trehalose and trimethylamine-N-oxide, can also be used for protein stabilization at a moderately high temperature or in the presence of denaturants. The ligand binding of proteins often enhances stability by virtue of coupling of binding with unfolding equilibrium. For instance, binding of biotin to streptavidin and anilinonaphthalene sulphonate derivatives to bovine serum albumin increased Tm values of these proteins. The effect of calcium binding on stability of serine protease, subtilisin S41 from the Antarctic Bacillus, also has been reported.
Improvements in stability also have been accomplished by chemical modification and immobilization. Chemical modification and immobilization have been used for improving protein stability by reducing conformational flexibility. For example, glycosidation of phenylalanine dehydrogenase with cyclodextrin derivatives enhanced its stability. Immobilization of penicillin G acylase on glyoxyl-agarose supports via lysine-mediated coupling improved its stability. In addition, reduced conformational flexibility can also be achieved by cross-linking the N- and C-termini of a target protein. For instance, beta lactamase and dihydrofolate reductase with their respective N- and C-termini connected through backbone cyclization were slightly more stable than the wild-type ones.
Previous methods of stabilizing proteins do have limitations. Enhanced stabilization achieved by rational, combinatorial and data-driven design involves changes in residues of a target protein usually in the form of mutations and recombinations. These changes very often compromise intrinsic properties of proteins, such as activity and specificity. This also occurs with chemical modification of proteins and their immobilization. Reduced conformational flexibility by modification and immobilization usually result in the significant loss of enzymatic activity. Recently, comprehensive directed evolution studies have demonstrated that stability and activity are not always inversely correlated. For instance, directed evolution of phosphate dehydrogenase led to identification of the variant with improved stability and activity. However, mutations and recombinations that improve stability with no compromise in activity or specificity are very rare and difficult to predict. This limitation would be even worse for stabilization of proteins with discontinuous catalytic domains. Mutation of residues to those commonly found in naturally existing stable counterparts improved stability of mesophilic proteins with no activity loss. However, only a small fraction of thermophilic proteins in nature have been identified. Also, a thermophilic protein with desired properties (such as activity and selectivity) is not always available from naturally existing ones. Employment of chaperones for stabilization is not very practical due to their lack of specificity and requirement of a relatively large dose. Stabilization by ligand addition requires tight binding (or the presence of excess ligands), which is not always available in normal proteins.
Therefore, it can be seen that new methods for the stabilization of proteins can be advantageous. It also can be seen that new methods for the stabilization of proteins that without modification of their primary sequence can be advantageous. The present invention is directed to such new methods and others.